title: The N-terminal membrane domain of yeast NADPH-cytochrome P450 (CYP) oxidoreductase is not required for catalytic activity in sterol biosynthesis or in reconstitution of CYP activity.
abstract: The disruption of Saccharomyces cerevisiae NADPH- cytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2UH, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Venkateswarlu
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
authors.forename: N J
authors.initials: NJ
authors.lastname: Manning
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
mesh_terms: D002384:Catalysis; D003001:Cloning, Molecular; D004875:Ergosterol / Q000096:biosynthesis*; D005816:Genetic Complementation Test; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D009251:NADPH-Ferrihemoprotein Reductase; D004875:Ergosterol